The site of binding of dansyl chloride (DNS-C1) on human serum prealbumin (PA) has been determined by the method of affinity labeling. DNS-C1 binds to the Lys-15 of two of the four identical subunits of PA. It was also shown that thyroxine (T4) and DNS-C1 bind to the same sites on PA. Bromoacetyl-T4 also binds to a Lys residue of PA. Thyroxine-binding globulin (TBG) was purified using the method of affinity chromatography. The pure TBG was used for the radioimmunoassay of serum TBG levels in patients with hepatocellular carcinoma.